The present invention relates generally to systems and methods for selectively treating selected regions of an individual biological cell, and more particularly to systems and techniques utilizing laminar flow channel systems for such treatment.
Complex behavior of cells, for example mitosis, growth, movement, metabolism, differentiation, apoptosis, etc. reflect integration of processes occurring in separate micro domains. Investigation of such behaviors require methods for delivering reagents to and/or into cells with subcellular resolution. Currently available techniques now used for micro manipulation of cells, for example micro injection, manipulation using mechanical or optical systems, etc., can, in some instances, provide subcellular resolution, but suffer from various limitations.
For example, micro manipulation techniques, such as the use of optical tweezers (Ashkin, A. and Dziedzic, J. M., xe2x80x9cInternal cell manipulation using infrared laser traps,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 86, 7914-7918 (1989), can provide limited subcellular spatial resolution, but such techniques are limited in their molecular specificity. Microinjection techniques can provide molecular specificity; however, they lack spatial control due to the rapid diffusion of small molecules within the cell. In addition, techniques such as microinjection also require physical disruption of the cell plasma membrane in order to provide reagents to the interior of the cell.
Microfluidic systems utilizing a multi-component laminar flow stream have been employed to create microfluidic sensor systems. Such microfluidic sensor systems are described, for example in Weigl, B. H. and Yager, P., xe2x80x9cMicrofluidic Diffusion-based Separation and Detection,xe2x80x9d Science 283, 346-347 (1999). Kennis et al., xe2x80x9cMicro Fabrication Inside Capillaries Using Multi Phase Laminar Flow Patterningxe2x80x9d, Science, Vol. 285 (1999) describes the use of a laminar flow based microfluidic system for fabricating microstructures in capillaries. Takayama, et al., xe2x80x9cPatterning Cells and Their Environment Using Multiple Laminar Fluid Flows and Capillary Networks,xe2x80x9d Proc. Natl. Acad. of Sci. USA, Vol. 96 (1999) describes using similar laminar flow based microfluidic networks to facilitate the spatial patterning of cells on a substrate and to provide a selected fluid environment to cells attached to a substrate.
Laminar flow occurs when two or more streams having a certain characteristic (low Reynolds number) are joined into a single, multi-component stream, also characterized by a low Reynolds number, such that the components are made to flow parallel to each other without turbulent mixing. The flow of liquids in small capillaries often is laminar. For a discussion of laminar flow and a definitions of the Reynolds number, the reader is referred to any of a large number of treatises and articles related to the art of fluid mechanics, for example, see Kovacs, G. T. A., xe2x80x9cMicromachined Transducers Sourcebook,xe2x80x9d WCB/McGraw-Hill, Boston (1998); Brody, J. P., Yager, P., Goldstein, R. E. and Austin, R. H., xe2x80x9cBiotechnology at Low Reynolds Numbers,: Biophys. J, 71, 3430-3441 (1996); Vogel, S., xe2x80x9cLife in Moving Fluids,xe2x80x9d Princeton University, Princeton (1994); and Weigl, B. H. and Yager, P., xe2x80x9cMicrofluidic Diffusion-based Separation and Detection,xe2x80x9d Science 283, 346-347 (1999), each incorporated herein by reference.
Analytical chemical techniques have utilized laminar flow to control the positioning of fluid streams relative to each other. U.S. Pat. No. 5,716,852 (Yager et al.), describes a chemical sensor including a channel-cell system for detecting the presence and/or measuring the presence of analytes in a sample stream. The system includes a laminar flow channel with two inlets in fluid connection with the laminar flow channel for conducting an indicator stream and a sample stream into the laminar flow channel, respectively. The indicator stream includes an indicator substance to detect the presence of the analyte particles upon contact. The laminar flow channel has a depth sufficiently small to allow laminar flow of the streams and length sufficient to allow particles of the analyte to diffuse into the indicator stream to form a detection area.
U.S. Pat. No. 4,902,629 (Meserol et al.), discusses laminar flow in a description of apparatus for facilitating reaction between an analyte in a sample and a test reagent system. At least one of the sample and test reagent system is a liquid, and is placed in a reservoir, the other being placed in a capillary dimensioned for entry into the reservoir. Entry of the capillary into the reservoir draws, by capillary attraction, the liquid from the reservoir into the capillary to bring the analyte and test reagent system into contact to facilitate reaction.
A variety of references describe small-volume fluid flow for a variety of purposes. U.S. Pat. No. 5,222,808 (Sugarman et al.), describes a capillary mixing device to allow mixing to occur in capillary spaces while avoiding the design constraints imposed by close-fitting, full-volume mixing bars. Mixing is facilitated by exposing magnetic or magnetically inducible particles, within the chamber, to a moving magnetic field.
U.S. Pat. No. 5,300,779 (Hillman et al.), describes a capillary flow device including a chamber, a capillary, and a reagent involved in a system for providing a detectable signal. The device typically calls for the use of capillary force to draw a sample into an internal chamber. A detectable result occurs in relation to the presence of an analyte in the system.
International Patent Publication No. WO 97/33737, published Mar. 15, 1996 by Kim et al., describes modification of surfaces via fluid flow through small channels, including capillary fluid flow. A variety of chemical, biochemical, and physical reactions and depositions are described.
Typical prior art techniques employed for selectively treating single cells or supplying an active substance to the interior of a biological cell are unable to create long-term intracellular gradients, particularly of small molecules (e.g. those with molecular weights less than about 600 and having diffusion coefficients within the cell of more than about 10xe2x88x926 cm2/s). Microinjection studies and fluorescence recovery after photobleaching (FRAP) studies have shown that such small molecules will diffuse throughout the cytoplasm or myoplasm of a typical mammalian cell attached to a substrate (e.g., an attached mammalian cell having a maximum spread dimension of about 130 xcexcm) within seconds the intracellular distribution of the molecules will reach 95% of an equilibrium distribution (i.e., there will be no region within the interior of the cell having a concentration of the molecule differing from another region of the cell by more than about 5%) within about 2 to about 5 minutes, even in the presence of some reversible binding of the molecule to immobilized cellular components, which binding tends to decrease the apparent diffusion coefficient (e.g. see Mastro, A. M., Babich, M. A., Taylor, W. D., and Keith, A. D., xe2x80x9cDiffusion of small molecules in the cytoplasm of mammalian cells,xe2x80x9d Proc. Natl. Acad. Sci. USA, Vol. 81, 3414-3418 (1984); and Blatter, L. A. and Wier, W. G., xe2x80x9cIntracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2,xe2x80x9d Biophys. J, Vol. 58, 1491-1499 (1990)). Thus, such methods are not well suited for creating intracellular gradients of such molecule having long-term duration.
Bradke and Dotti, xe2x80x9cThe Role of Local Actin Instability in Axon Formation,xe2x80x9d Science, Vol. 283, 1999, describe a micropipetting technique for selectively treating a region of an axon of a neuron with a cytoskeletal disrupting substance. The technique described utilizes selectively positioned micropipettes to direct a flow of liquid containing the cytoskeletal disrupting substance such that it impinges upon a portion of the axon extending away from the main body portion of the cell. By using this technique, the actin cytoskeleton in the region of the axon upon which the fluid impinges can be selectively depolymerized. The technique described, however, is only able to create a flowing fluid over a portion of the cell, with the rest of the cell submerged in quiescent fluid. Also, the micropipetted fluid will have a tendency to undergo convective mixing with the quiescent fluid surrounding the cell, making the technique potentially poorly suited for selectively treating parts of the main body portion of the cell.
While the above and other references describe useful techniques for chemical, biochemical, and physical modifications of surfaces, analytical detection, and the treatment of single cells with desired substances, a need exists for improved, small scale systems and methods able to selectively treat parts of a single cell, including portions of a main body portion of a single cell, and able to establish long-term gradients of active substances within subcellular regions of a single cell.
The present invention is directed, in certain embodiments, to improved, small scale systems and methods able to selectively treat parts of a single cell, including, in certain embodiments, portions of a main body portion of a single cell, and able, in certain embodiments, to establish long-term gradients of active substances within subcellular regions of a single cell. The present invention provides, in some embodiments, techniques for selectively contacting a portion of the surface of a biological cell with a fluid or fluid component carrying a particular potential for a biophysical or biochemical interaction with the cell, and simultaneously contacting a different portion of the surface of the cell with another fluid or fluid component having a different potential for the biophysical or biochemical interaction with the cell.
In one aspect, a method is disclosed, the method comprising establishing a flowing stream of a fluid against a surface of a cell, the stream including at least first and second components in contact with first and second portions of the cell, respectively. The first component of the stream includes therein, at a first concentration, a substance able to bind to the surface of the cell, permeate across the cell plasma membrane into the interior of the cell, or both. The second component of the stream has a second concentration of the substance therein. The method further comprises binding the substance to the surface of the first portion of the cell, permeating the substance across the cell plasma membrane of the first portion of the cell, or both, to an extent different than that at the second portion of the cell.
In another embodiment, a method is disclosed, the method comprising selectively providing to a first portion of the exterior of a cell a first flowing fluid containing a substance able to effect a biochemical or biophysical interaction within the cell. The method further comprises selectively providing to a second portion of the exterior of the cell a second flowing fluid removing from the second portion of the exterior of the cell said substance, thereby establishing within the cell a gradient of an active substance.
In another embodiment, a method is disclosed, the method comprising selectively exposing a first portion of the exterior of a cell to a first fluid containing a substance able to effect a biochemical or biophysical interaction within the cell, the first portion of the exterior of the cell comprising a portion of a main body of the cell, and selectively exposing a second portion of the exterior of the cell to a second fluid removing from the second portion of the exterior of the cell said substance, thereby establishing within the cell a gradient of an active substance. The gradient being characterized by the existence of a first region within the cell, proximate to at least a portion of the first portion of the exterior of the cell, having a first concentration of the active substance and the existence of a second region within the cell, proximate to at least a portion of the second portion of the exterior of the cell, having a second concentration of the active substance, the first concentration of the active substance differing from the second concentration of the active substance by at least about 5% at a time exceeding about 5 min after the cell was first exposed to the first and second fluids.
In yet another embodiment, a method is disclosed, the method comprising establishing within a cell a gradient of a freely diffusable active substance, characterized by the existence of a first region within the cell having a first concentration of the active substance and the existence of a second region within the cell having a second concentration of the active substance, the first concentration of the active substance differing from the second concentration of the active substance by at least about 5% at a time exceeding about 5 min after the commencement of the establishment of the gradient.
In yet another embodiment, a method is disclosed, the method comprising creating a first region within a cell of a selected cell type, the first region containing freely diffusable active substance, the first region comprising a portion of a main body of the cell. The method further comprises creating a second region within the cell essentially free of freely diffusable active substance. The method also involves detecting, for each of the first and second regions, at least one parameter indicative of a response of the cell to the active substance determinative of the efficacy of a treatment with the active substance on the cell type.
In another embodiment, a method is disclosed, the method comprising allowing a substance to bind to a first region of the exterior of a cell membrane of a selected cell type and creating a second region of the exterior of the cell membrane that is essentially free of the bound substance. The method also involves detecting, for each of the first and second regions, at least one parameter indicative of a response of the cell to the bound substance determinative of the efficacy of a treatment with the substance on the cell type.
In yet another embodiment, a method is disclosed, the method comprising selectively providing to a first portion of the plasma membrane of a cell a first flowing fluid containing therein a substance, which is able to permeate across the plasma membrane, at a concentration exceeding or equal to a maximum concentration of the substance within the cell, and selectively providing to a second portion of the plasma membrane of the cell a second flowing fluid containing therein a concentration of the substance, which is able to permeate across the plasma membrane, less than or equal to a minimum concentration of the substance within the cell.
In another embodiment, a method is disclosed, the method comprising establishing a flowing stream of a fluid against a surface of a cell, the stream including at least first, second and third components in contact with first, second, and third portions of the cell, respectively, the second component of the stream being interposed between the first component of the stream and the third component of the stream. The first component of the stream and the third component of the stream each carry a different potential for a biophysical or biochemical interaction with the cell than the second component of the stream. The method further involves carrying out the biophysical or biochemical interaction at the first and third portions of the cell to an extent different than at the second portion of the cell.
In yet another embodiment, a method is disclosed, the method comprising establishing a flowing stream of a fluid, the stream including at least first and second components adjacent to each other and defining therebetween a boundary. The method further includes carrying out a biophysical or biochemical interaction at a first portion of a cell proximate the boundary selectively, to an extent different than at a second portion of the cell.
In another aspect, an article is disclosed. The article comprises a substrate having at least one cell positioned on a surface of the substrate and a flowing fluid stream in contact with the surface. The stream includes at least first and second components in contact with first and second portions of the cell, respectively. The first component of the stream included therein at a first, essentially uniform concentration a substance able to bind to an exterior surface of the cell, permeate across the cell membrane into the interior of the cell, or both. The second component of the stream has a second, essentially uniform concentration of the substance therein.
Other advantages, novel features, and objects of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings, which are schematic and which are not intended to be drawn to scale. In the figures, each identical or nearly identical component that is illustrated in various figures is represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.